Synthetic biology is seen as an emerging discipline for engineering cells more easily and predictably. Several advances have made this possible, such as the increasing numbers of sequenced genomes as raw materials, rapid synthesis of large DNAs, the ability to quantitate the inner workings of single cells, the potential to reprogra entire organisms with new genomes, and a vision that biological design has some analogy to logic circuits. However, myriad studies on cell organization add a degree of complexity and regulation that has not been appreciated in the overall design processes. The overarching goal of this proposal is to harness the multidimensionality of biology to optimize the production of novel pathways, complexes, and cells. A limitation of current synthetic biology is the tendency to engineer biological systems as if they function in a linear, digital computer-like manner. However, cells function in three spatial dimensions and over time, using multi-dimensionality in the form of protein/nucleic acid/membrane complexes and organelles. The proposed work focuses on assembling, in a predictable manner, protein complexes that move in more than one dimension and on different time scales. This work thus encompasses a new direction that will integrate synthetic biology with cell and structural biology, with direct health-relatedness throug its broader implications for development of protein and cell-based therapeutics. The Specific Aims are to: 1) construct memory circuits that record different levels of cytokine signaling; 2) determine the quantitative and spatial requirements for information transmission between the cell surface and the nucleus; and 3) simulate the behavior of proteins that signal from the cell surface to the nucleus. Type I Interferon/STAT signaling will be studied, because it has therapeutic significance, a high signal-noise ratio, and many questions remain about signaling mechanisms. Multi-element gene-based memory circuits that record for later inspection key events in the existence of a cell will be implemented. Prototype targeted cytokines for activity in cell-based assays will be developed; these will be designed to represent synthetic biological constructs and engineered protein therapeutics, as well as natural flexible proteins that use multiple weak interactions to accomplish complex assembly tasks. Finally, a simulation system that will model the spatiotemporal behavior of flexible multi-domain proteins typical of synthetic-biological constructs or candidate therapeutics will be implemented.